Studies on Methylmalonyl-CoA Mutase from Escherichia coli

Methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2), a coenzyme B12-dependent

enzyme, catalyses the inter conversion of succinyl-CoA and methylmalonyl-

CoA. The gene (sbm) encoding this enzyme is found in Escherichia coli (E. coli)

at 62.3min on the E. coli chromosome. However, the metabolic role of this

enzyme in the organism is not known. This project involves an investigation into

this metabolic obscurity.

The sbm gene is part of a four gene operon which also includes argK (or ygfD)

that codes for a protein kinase catalysing the phosphorylation of two periplasmic

binding proteins involved in cationic amino acid transport, ygfG that codes for

methylmalonyl-CoA decarboxylase and ygfH that codes for propionyl-CoA:

succinyl-CoA transferase. From existing literature we suspect that this operon,

including the sbm gene, could be involved in the utilisation of unusual carbon

sources such as succinate and propionate. An insertion mutant of the sbm gene

created by transposon mediated mutagenesis was used for investigating the role

of this gene. The wild type E. coli K12 strain, E. coli TR6524 and the mutant E.

coli K12 (sbm::MudJ) were used in this study.

Growth of the two strains (E. coli TR6524 and FA1P1) in minimal media with

three different concentrations (0.05, 0.5, 5.0μg/mL) of vitamin B12 and in the

presence succinate, propionate or glucose as the sole source of carbon, was

studied. Growth was typical in media with glucose with no major differences in

the growth pattern of the wild type and mutant strain. However, the two strains

exhibited a differential growth pattern in media containing succinate, with the

wild type growing faster than the mutant, indicating the role of the sbm gene in

the utilisation of this carbon source. Growth in media containing propionate as

the sole carbon source indicated only marginal differences in the growth pattern

of the wild type and mutant strain. This result possibly suggests that the other

pathways for propionate utilisation in E. coli compensate for the lack of a

functional Sbm protein in the mutant strain. Promoter analysis indicated the presence of a promoter induced by σS, a

transcription factor involved in the expression of proteins under stress or

stationary phase growth conditions. Reverse transcription polymerase chain

reaction (RT-PCR) studies of the genes of the sbm operon (sbm-argK-ygfGygfH)

under the same growth conditions were carried out. Densitometric analysis

of the PCR products suggested that the transcription level of sbm was higher in

E. coli grown in succinate as compared to when grown in glucose and not as

much when grown in propionate indicating a transcriptional level control of the

sbm gene expression during the utilisation of succinate. RT-PCR studies also

indicated a higher level of transcription of the gene in the stationary phase of the

culture during the utilisation of succinate. Real time reverse transcription PCR

(QPCR) analysis was used for the absolute quantification of the transcription of

the genes of the sbm operon. An increase in the mRNA levels corresponding to

the sbm, argK and ygfG genes was observed as E. coli TR6524 growth reached

stationary phase, in the presence of succinate or propionate as the sole source of

carbon as compared to glucose, In contrast, the highest mRNA levels

corresponding to the ygfH gene were observed in the early log-phase of growth.

This indicated a differential transcriptional level control of the genes within the

operon. This study further established the possible role of this operon in the

utilisation of succinate and propionate.

The MCM enzyme activity measurement in the whole cell extracts of the wild

type E. coli K12, grown under the above mentioned conditions, led to the first

ever measurement of MCM activity in wild type E. coli. These measurements

also revealed a four fold increase of the MCM specific activity in the case of

growth in succinate (4.76x10-3U/mg) and a two fold increase for growth in

propionate (2.79x10-3U/mg) compared to that observed with growth in glucose

(1.37x10-3U/mg), indicating a significant level of involvement of the enzyme in

succinate utilisation, and to a lesser extent in propionate utilisation.

The proteomic analysis to understand the gene expression pattern of E. coli

TR6524 was carried out using cells harvested at the stationary phase. The results

showed that growth conditions induced the expression of transport related (HisJ,

DppA) and energy generating proteins (PckA, AceF) required by E. coli to cope with the stressful growth conditions. However, Sbm was not identified among

the limited protein spots that were analysed.

Finally, E. coli K12 sbm gene was successfully cloned into B. cereus SPV

leading to the development of a metabolically engineered polyhydroxyalkanoate

producing strain of B. cereus. The intention was to provide the bacteria with a

natural intracellular source of propionyl-CoA, leading to the production of the

P(3HB-co-3HV) copolymer from structurally non related carbon sources like

glucose.

Hence, this work has initiated investigation into the metabolic role of the sbm

gene product in E. coli. In addition, it has also led to the use of this gene product

in metabolic engineering applications.